This invention relates generally to enzyme inhibitors, and particularly to a new class of peptide inhibitors of proteolytic enzymes.
Proteases are enzymes which cleave proteins at single, specific peptide bonds. Cuypers, et al., J. Biol Chem. 257:7086 (1982), and the references cited therein, classify proteases on a mechanistic basis into four classes: serine, cysteinyl or thiol, acid or aspartyl, and metalloproteases. Members of each class catalyze the hydrolysis of peptide bonds by a similar mechanism, have similar active site amino acid residues, and are susceptible to class-specific inhibitors. For example, all serine proteases that have been characterized have an active site serine residue, and are susceptible to inhibition by organofluorophosphates and substrate-derived chloromethyl ketones. Metalloproteases are inhibited by chelating agents. The reactivity of more specialized inhibitors with individual proteases is, however, highly dependent upon the structure of the inhibitor, and, in the case of peptide inhibitors, upon amino acid sequence.
Lienhard, in Enzyme Inhibitors as Drugs, Sandler, ed., (University Park Press, Baltimore, 1980) pp. 43-51, suggests that boronic acid analogous (I) of certain amino acids and peptides are likely transition-state analogues, and potentially "extremely potent inhibitors" of serine and thiol proteases. ##STR1##
Koehler, et al., Biochemistry 10:2477 (1971), disclose inhibition of chymotrypsin, a serine protease, by 2-phenylethaneboronic acid (K.sub.i =2.9 mM). Matteson, et al., J. Am. Chem. Soc. 103:5241 (1981) describe preparation of (R)-1-acetamido-2-phenylethaneboronic acid (II) and its use as an inhibitor of chymotrypsin (K.sub.i =4 .mu.M). ##STR2## Lindquist, et al., Arch. Biochem. Biophys. 160:135 (1974), describe use of 2-phenylethaneboranic acid and benzeneboronic acid as inhibitors of subtilisin, a bacterial chymotrypsin-like serine protease. Lindquist, et al., J. Am. Chem. Soc. 99: 6435 (1977), disclose synthesis of N-benzoylaminomethaneboronic acid, and use of the latter compound as a potent, competitive inhibitor of .alpha.-chymotrypsin (K.sub.i =1-4 .mu.M). However, Matteson, et al., J. Am. Chem. Soc. 103:5241 (1981) suggest that the compound actually obtained by Lindquist, et al., was more likely an isomer, the imido ester III. ##STR3##
Aberrant proteolysis has been associated with disease states in man and experimental animals, notably emphysema. Barrett, in Enzyme Inhibitors as Drugs, Sandler, ed., (University Park Press, Baltimore, 1980) pp. 216-229, and the references cited therein, suggests possible contributory roles for the human neutrophil serine proteases, elastase and cathepsin G, in a diverse group of disease states marked by proteolysis and consequent tissue destruction. Included are rheumatoid arthritis, corneal ulcers, and glomerular nephritis. Further, the tissue damage resulting from bacterial and other parasitic infections may be attributable to the activity of serine proteases of pathogenic origin.
Emphysema is defined by the American Thoracic Society as "an anatomic alteration of the lung characterized by an abnormal enlargement of the air spaces distal to the terminal nonrespiratory bronchiole, accompanied by destructive changes of the alveolar walls." In all forms of emphysema the elastic fibers of alveolar walls are disrupted. Considerable evidence has been accumulated for the role of proteolysis in disruption of elastin fibers. Furthermore, this aberrant proteolysis has been ascribed to a leukocyte serine protease, neutrophil elastase. Neutrophil elastase is readily accessible to lung tissue, since neutrophils are sequestered in lung capillaries and connective tissue in response to lung irritants.
Janoff, in Molecular Basis of Biological Processes, Berlin, et al., eds., (Academic Press, New York, 1978) pp. 225-259, reviews evidence for the role of neutrophil elastase in pathogenesis of emphysema. Emphysema can be induced in experimental animals by intrapulmonary administration of certain proteases. The severity of the disease is directly correlated with the elastolytic titer of the protease mixture instilled, while enzymes which do not hydrolyze elastin are ineffective. Laurell, et al., Scand. J. Clin. Lab. Invest., 15:132, (1963), and Tobin, et al., Br. J. Dis. Chest 77:14 (1983), correlate .alpha..sub.1 -antitrypsin (.alpha..sub.1 -protease inhibitor) deficiency with incidence of emphysema. Further, Thompson, TIBS 7:349 (1982), reports that 40% of individuals deficient in .alpha..sub.1 -antitrypsin die of lung disease. .alpha..sub.1 -Antitrypsin is a major protease inhibitor in plasma, and effectively inhibits neutrophil elastase, exhibiting a K.sub.assoc. of 6.5.times.10.sup.7 M.sup.-1 s.sup.-1.
In a continuing search for therapeutic agents useful for treatment of emphysema, several groups have prepared and tested inhibitors of neutrophil elastase. Powers, et al., Biochim. Biophys. Acta 485:156 (1977), tested a number of chloromethyl ketone peptide analogues for inhibitory capacity. Of the compounds tested, N-methoxysuccinyl-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethyl ketone (MeOSuc-Ala-Ala-Pro-ValCH.sub.2 Cl) was the most effective inhibitor of neutrophil elastase, exhibiting inhibitory activity at a concentration of 25 .mu.M. A related peptidyl chloromethyl ketone, N-acetyl-L-alanyl-L-alanyl-L-prolyl-L-alanine chloromethyl ketone (Ac-Ala-Ala-Pro-AlaCH.sub.2 Cl) was reported by Kleinerman, et al., Am Rev. Resp. Dis. 121:381 (1980) to effectively block experimentally-induced emphysema in hamsters when administered intraperitoneally. MeOSuc-Ala-Ala-Pro-ValCH.sub.2 Cl was determined by Janoff, et al., Am Rev. Resp. Dis 121:1025 (1980) to be an effective agent in the treatment of emphysema in mice when given orally. N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethyl ketone (Suc-Ala-Ala-Pro-ValCH.sub.2 Cl) was effective in hamsters following intratracheal administration, according to Stone, et al., Am. Rev. Resp. Dis. 124:567 (1981). However, as Janoff, et al., supra, note, many questions regarding the toxicity, immunogenicity, and carcinogenicity of chloromethyl ketones, which are alkylating agents, remain unresolved. Furthermore, the rapid loss of effectiveness of this class of compounds following administration complicates their use as therapeutic agents. For example, the most effective inhibitor tested by Janoff, MeOSuc-Ala-Ala-Pro-ValCH.sub.2 Cl, was ineffective if administered to mice more than fifteen minutes prior to challenge by intrapulmonary instillation of elastase.
Other inhibitors of elastase which are reported in the chemical and medical literature include certain aza peptides, disclosed by Powers, et al., Biochem. Biophys. Res. Comm. 67:639 (1975); trifluoroacetyl peptide chloromethyl ketones, disclosed by Lestienne, et al., J. Biol. Chem. 254:5219 (1979); certain heterocyclic species disclosed by Teshima, et al., J. Biol. Chem. 257:5085 (1982); arylsulfonyl fluorides, described by Yoshimura, et al., J. Biol. Chem. 257:5077 (1982); trifluoromethylated dipeptides of formula (IV), disclosed in U.K. Patent Application 2,040,291A; and certain 2-pyridyl-1,2-benzisothiazolinone-1,1-dioxide compounds, disclosed in Jones et al., U.S. Pat. No. 4,369,183. ##STR4##
Accordingly, new classes of potent inhibitors of serine and other proteases, characterized by longlasting inhibitory capacity and low mammalian toxicity, are potentially valuable therapeutic agents for treatment of mammalian proteolytic disease states. notably emphysema.